This is an application for four years of support for a proposal to elucidate the functional role of Na+/H+ exchanger proteins in normal salivary gland physiology. Na+/H+ exchange is the ubiquitous and primary mechanism for regulating the intracellular pH in virtually all mammalian cells. Up-regulation of Na+/H+ exchangers buffers the stimulation-induced acidification and increases the fluid secretion, whereas inhibition of this exchanger dramatically reduces the stimulated salivary flow rate. The kinetic and regulatory properties of Na+/H+ exchange in sublingual and parotid acinar cells are distinct and are hypothesized to reflect either the dominance of different Na+/H+ exchanger isoforms or distinct regulatory mechanisms are at work in these two cell types. To distinguish between these possibilities, the investigators will examine the kinetic and regulatory properties of Na+/H+ exchange and determine the tissue expression, localization and the functional significance of Na+/H+ exchangers in sublingual and parotid acinar cells. Four Specific Aims are identified: Aim 1 is to examine the intracellular pH (pHi) "set point", Na+ dependency, and inhibitor sensitivity of Na+/H+ exchange in sublingual and parotid acinar cells. The results of Aim 1 are intended to aid in identifying the dominant Na+/H+ exchanger present and its role in salivary gland function; Aim 2 is to assess whether changes in cell volume, cAMP or Ca2+ regulate Na+/H+ exchange by monitoring pHi and Na+ fluxes. The results from these efforts are to aid in identifying the Na+/H+ exchanger isoform and in determining its properties in different functional states; Aim 3 is to examine the transcript size and expression level of the four different Na+/H+ exchanger isoforms found by PCR analysis. This will be done by Northern blot and ribonuclease protection assays. Additional efforts are planned to determine whether the four different Na+/H+ exchanger isoforms are differentially expressed among sublingual and parotid salivary glands, using antibodies generated against specific peptides. Using confocal microscopy, the applicant will visualize the subcellular and cell-type-specific location of the Na+/H+ exchangers; Aim 4 is to determine the physiological role of Na+/H+ exchange in vivo and in vitro by assessing the effect of inhibition on salivary flow rate, and on intracellular pH regulation, at the single cell level. The results of Aim 4 are intended to verify the role of Na+/H+ exchanger membrane proteins during stimulation of the sublingual and parotid acinar cells.